Dual-target biosensor cell assays

ABSTRACT

A method and apparatus, as defined herein, for use in compound screening, compound profiling, or both assays, for example, against two different cellular targets in, for example, a single cell-type.

CLAIMING BENEFIT OF PRIOR FILED U.S. APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/068,266, filed on Mar. 5, 2008. The content of this document andthe entire disclosure of publications, patents, and patent documentsmentioned herein are incorporated by reference.

BACKGROUND

The disclosure relates to biosensors, such as resonant waveguide grating(RWG) biosensors and electric impedance biosensors, for use in cellassay applications, for example, for compound screening and compoundprofiling.

SUMMARY

The disclosure provides a method and apparatus for use in cell assaysfor compound screening and compound profiling, for example, against twodifferent cellular targets in a single cell-type, or against twodifferent cellular targets, each of which is in a distinct cell-type.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show aspects of biosensor-based cell assays for dual- orduplexed-target specific screening using a RWG biosensor and electricbiosensor, respectively, in embodiments of the disclosure.

FIGS. 2A-2D show an exemplary RWG biosensor-based cell assays againsttwo classes of G protein-coupled receptors (GPCRs), in embodiments ofthe disclosure.

FIGS. 3A-3C show another exemplary RWG biosensor-based cell assayagainst two classes of G protein-coupled receptors, in embodiments ofthe disclosure.

FIG. 4 shows results of an exemplary duplexed and target-specific screenusing an RWG biosensor, in embodiments of the disclosure.

FIGS. 5A-5C show results of exemplary optical biosensor cellular assaysfor two endogenous receptors in A431 cells—β2-adrenergic receptor andhistamine H1 receptor, in embodiments of the disclosure.

FIGS. 6A-6D show results of an exemplary duplexed and target-specificscreen using an RWG biosensor, in embodiments of this disclosure.

FIGS. 7A-7F show results of an exemplary compound library screening foragonists using biosensor cellular assays, in embodiments of thisdisclosure.

FIG. 8 shows results of an exemplary compound library screening foragonists using biosensor cellular assays, in embodiments of thisdisclosure.

FIGS. 9A-9B show results of an exemplary compound library screening forantagonists using biosensor cellular assays, in embodiments of thisdisclosure.

DETAILED DESCRIPTION

Various embodiments of the disclosure will be described in detail withreference to drawings, if any. Reference to various embodiments does notlimit the scope of the invention, which is limited only by the scope ofthe claims attached hereto. Additionally, examples in this specificationare not limiting and merely set forth some of the many possibleembodiments for the claimed invention.

Definitions

“Assay,” “assaying” or like terms refers to an analysis to determine,for example, the presence, absence, quantity, extent, kinetics,dynamics, or type of a cell's optical or bioimpedance response uponstimulation with an exogenous stimuli, such as a ligand candidatecompound.

“Attach,” “attachment,” “adhere,” “adhered,” “adherent,” “immobilized”,or like terms generally refer to immobilizing or fixing, for example, asurface modifier substance, a compatibilizer, a cell, a ligand candidatecompound, and like entities of the disclosure, to a surface, such as byphysical absorption, chemical bonding, and like processes, orcombinations thereof. Particularly, “cell attachment,” “cell adhesion,”or like terms refer to the interacting or binding of cells to a surface,such as by culturing, or interacting with cells with a surface, such asa biosensor surface. The biosensor surface can be unmodified ormodified, such as having a surface coating, an anchoring material, acompatibilizer (e.g., fibronectin, collagen, lamin, gelatin, polylysine,etc.), or like modifications that promote cell adhesion and cell statusor growth. For suspension cells, the cells can be, for example, broughtto contact with the detection zone of the biosensor through physicalsettlement during incubation, or through surface-cell interactions. Thesurface-cell interactions can be achieved by several means, e.g.,covalently coupling of reactive surfaces with the basal cell membraneproteins or molecules, charge-based electrical interactions, binding ofthe sensor surface-presenting molecules (e.g., antibody, ligand) withthe basal cell surface molecules, or like approaches.

“Adherent cells” refers to a cell or a cell line or a cell system, suchas a prokaryotic or eukaryotic cell, that remains associated with,immobilized on, or in certain contact with the outer surface of asubstrate. Such type of cells after culturing can withstand or survivewashing and medium exchanging process, a process that is prerequisite tomany cell-based assays. “Weakly adherent cells” refers to a cell or acell line or a cell system, such as a prokaryotic or eukaryotic cell,which weakly interacts, or associates or contacts with the surface of asubstrate during cell culture. However, these types of cells, forexample, human embryonic kidney (HEK) cells, tend to dissociate easilyfrom the surface of a substrate by physically disturbing approaches suchas washing or medium exchange. “Suspension cells” refers to a cell or acell line that is preferably cultured in a medium wherein the cells donot attach or adhere to the surface of a substrate during the culture.“Cell culture” or “cell culturing” refers to the process by which eitherprokaryotic or eukaryotic cells are grown under controlled conditions.“Cell culture” not only refers to the culturing of cells derived frommulticellular eukaryotes, especially animal cells, but also theculturing of complex tissues, organs, or like systems.

“Cell” or like term refers to a small usually microscopic mass ofprotoplasm bounded externally by a semipermeable membrane, optionallyincluding one or more nuclei and various other organelles, capable aloneor interacting with other like masses of performing all the fundamentalfunctions of life, and forming the smallest structural unit of livingmatter capable of functioning independently including synthetic cellconstructs, cell model systems, and like artificial cellular systems.

“Cell system” or like term refers to a collection of cells and caninclude more than one type of cells (or differentiated forms of a singletype of cell), which interact with each other, thus performing abiological or physiological or pathophysiological function. Such cellsystem can include, for example, an organ, a tissue, a stem cell, adifferentiated hepatocyte cell, and like cell systems.

“Target” or like term refers to a cellular protein or biomolecule whoseactivation can mediate cell signaling or modulate cellular functions. Atarget can be, for example, a receptor, a phosphatase, a kinase, anenzyme, a DNA, an RNA, and like entities. A receptor can be, forexample, a G protein-coupled receptor (GPCR), a receptor tyrosine kinase(RTK), a transporter, an ion channel, an integrin receptor, asodium/proton exchanger, and like entities. A kinase can be, forexample, protein kinase A, protein kinase C, mitogen-activated protein(MAP) kinases, an extracellular signal-regulated kinases, Src, Rhokinase, focal adhesion kinase, and like entities. An enzyme can be, forexample, a membrane-bound adenylyl cyclase, a soluble adenylyl cyclase,a protease, and like entities.

“Dual-,” “duplex,” “duplexed-,” or like terms refers to an assay thatmeasures the cellular responses or activities mediated through twodistinct targets, for example, a G_(q)-coupled receptor and aG_(s)-coupled receptor, two different G_(q)-coupled receptors, a GPCRand a receptor tyrosine kinase, a GPCR and an enzyme, a GPCR and akinase, and like combinations.

“Multiplex,” “multiplexed,” or like terms refers to an assay thatmeasures the cellular responses or activities mediated through more thantwo individual targets. The targets can belong to, for example, a sameclass of targets, for example, GPCRs, or different classes of targets,for example, two GPCRs and one receptor tyrosine kinase.

“Screen,” “screening,” or like terms refers to, for example, asystematic survey of one or more compounds or drug candidates orbiologicals (e.g., RNAi, antibody) to examine their pharmacologicalactivities acting on a particular target, a cell type, or a cell system.Pharmacological or biological activity is an expression describing thebeneficial or adverse effects of a drug on living matter.

“Profile,” “profiling,” or like terms refers to an extrapolation ofinformation about pharmacological activity of a drug candidate, acompound, or a biological acting on a living cell or cell system throughone or more cellular targets, based on a known or predetermined signaloutput, such as the amplitude of an optical or bioimpedance response ofcells, mediated through a particular target.

“Marker” or like term refers, for example, to a molecule, a biomolecule,or a biological that is able to modulate the activities of at least onecellular target (e.g., a G_(q)-coupled receptor, a G_(s)-coupledreceptor, a G_(i)-coupled receptor, a G_(12/13)-coupled receptor, an ionchannel, a receptor tyrosine kinase, a transporter, a sodium-protonexchanger, a nuclear receptor, a cellular kinase, a cellular protein,etc.), and result in a reliably detectable biosensor output as measuredby a biosensor. Depending on the class of the intended cellular targetand its subsequent cellular event(s), a marker could be an activator,such as an agonist, a partial agonist, an inverse agonist, for example,for a GPCR or a receptor tyrosine kinase or an ion channel or a nuclearreceptor or a cellular enzyme adenylate cyclase. The marker could alsobe an inhibitor for certain classes of cellular targets, for example, aninhibitor or a disruptor for actin filament, or microtubule, or aninhibitor for a kinase such as Rho kinase, or an antibody, or likeentities for a cell surface molecule, such as anti-epidermal growthfactor receptor antibody.

“Detect” or like term refers to an ability of the apparatus and methodsof the disclosure to discover or sense, simultaneously, at least twoligand-induced cellular responses, and to distinguish the sensedresponses from an absence of the ligand compound.

“Identify” or like term refers to an ability of the apparatus andmethods of the disclosure to not only recognize a ligand compound'simpact on at least two targets but to also classify the nature of theligand compound's impact or interaction on at least two targets.

“Stimulus,” “therapeutic candidate compound,” “therapeutic candidate,”“prophylactic candidate,” “prophylactic agent,” “ligand candidate,” orlike terms refer to a molecule or material, naturally occurring orsynthetic, which is of interest for its potential to interact with acellular target immobilized or attached to the biosensor. A therapeuticor prophylactic candidate can include, for example, a chemical compound,a biological molecule, a peptide, a protein, a biological sample, a drugcandidate small molecule, a drug candidate biologic molecule, a drugcandidate small molecule-biologic conjugate, and like materials ormolecular entity, or combinations thereof, which can specifically bindto or interact with at least one of two or more cellular targets such asa protein, DNA, RNA, an ion, a lipid, or like structure or component ofa live-cell.

“Biosensor” or like term refers to an article, that in combination withappropriate apparatus, can detect a desired analyte. A biosensor cancombine a biological component with a physicochemical detectorcomponent. A biosensor can typically consist of three parts: abiological component or element (such as tissue, microorganism,pathogen, cells, cell component, or combinations thereof), a detectorelement (operating in a physicochemical way such as optical,piezoelectric, electrochemical, thermometric, magnetic, or like manner),and a transducer associated with both components. In embodiments, theoptical biosensor can convert a molecular recognition or molecularstimulation event in a live-cell into a quantifiable signal.

Abbreviations, which are well known to one of ordinary skill in the art,may be used (e.g., “h” or “hr” for hour or hours, “g” or “gm” forgram(s), “mL” for milliliters, and “rt” for room temperature, “nm” fornanometers, and like abbreviations).

“Include,” “includes,” or like terms means including but not limited to.

“About” modifying, for example, the quantity of an ingredient in acomposition, concentrations, volumes, process temperature, process time,yields, flow rates, pressures, and like values, and ranges thereof,employed in describing the embodiments of the disclosure, refers tovariation in the numerical quantity that can occur, for example, throughtypical measuring and handling procedures used for making compounds,compositions, concentrates or use formulations; through inadvertenterror in these procedures; through differences in the manufacture,source, or purity of starting materials or ingredients used to carry outthe methods; and like considerations. The term “about” also encompassesamounts that differ due to aging of, for example, a composition,formulation, or cell culture with a particular initial concentration ormixture, and amounts that differ due to mixing or processing acomposition or formulation with a particular initial concentration ormixture. Whether modified by the term “about” the claims appended heretoinclude equivalents to these quantities.

“Consisting essentially of” in embodiments refers, for example, to acomposition, a method of making or using a composition, formulation, orcomposition on the surface of the biosensor, and articles, devices, orapparatus of the disclosure, and can include the components or stepslisted in the claim, plus other components or steps that do notmaterially affect the basic and novel properties of the compositions,articles, apparatus, and methods of making and use of the disclosure,such as particular reactants, particular additives or ingredients, aparticular agents, a particular cell or cell line, a particular surfacemodifier or condition, a particular ligand candidate, or like structure,material, or process variable selected. Items that may materially affectthe basic properties of the components or steps of the disclosure or mayimpart undesirable characteristics to the present disclosure include,for example, decreased affinity of the cell for the biosensor surface,decreased affinity of the ligand candidate for a cell, decreasedaffinity of a pathogen for a cell, anomalous or contrary cell activityin response to a ligand candidate or like stimulus, and likecharacteristics. In some instances, the foregoing examples ofundesirable characteristics can instead be highly desirable andbeneficial in screening or profiling applications of the presentdisclosure, such as discovery of conditions or ligands that decrease theaffinity of the ligand candidate for a cell, or decrease the affinity ofa pathogen for a cell.

The indefinite article “a” or “an” and its corresponding definitearticle “the” as used herein means at least one, or one or more, unlessspecified otherwise.

Specific and preferred values disclosed for components, ingredients,additives, cell types, pathogens, and like aspects, and ranges thereof,are for illustration only; they do not exclude other defined values orother values within defined ranges. The compositions, apparatus, andmethods of the disclosure include those having any value or anycombination of the values, specific values, more specific values, andpreferred values described herein.

The disclosure provides a method and apparatus for use in compoundscreening and compound profiling assays, for example, against twodifferent cellular targets in a single cell-type.

In embodiments, the disclosure provides a multiplexed andtarget-specific screening method for compound profiling, screening, orboth, using a label-free biosensor.

In embodiments, the screening method can comprise, for example:

providing a biosensor;

immobilizing a cell line sample expressing at least two differenttargets (e.g., a first target and a second target) onto the biosensorsurface;

contacting, such as incubating, the cell line sample contacted with thebiosensor with a ligand candidate for a time;

contacting, such as incubating, the ligand candidate-treated cell linewith a mixture containing at least two markers, each marker canselectively modulate the activity of at least one of the differenttargets;

monitoring the biosensor output during the incubations; and

determining the effect of the ligand candidate on the mixture-inducedbiosensor output.

In embodiments, the target can be, for example, at least one of: areceptor, a cellular protein, or a combination thereof. The cellularprotein can be, for example, at least one of a cellular enzyme, acellular kinase, a cellular structural protein, or a combinationthereof. The receptor can be, for example, at least one of aG_(q)-coupled receptor, a G_(s)-coupled receptor, a G_(i)-coupledreceptor, a G_(12/13)-coupled receptor, a receptor tyrosine kinase, anion channel, a sodium-proton exchanger, an integrin receptor, atransporter, or a combination thereof. In embodiments, the marker canbe, for example, at least one of an agonist, a partial agonist, or aninverse agonist. In embodiments, the agonist can, for example, activatea target and produce a detectable biosensor output signal. Inembodiments, the marker can be, for example, at least one of aninhibitor, or an antibody, where the marker can activate a target andproduce a detectable biosensor output signal. In embodiments, each ofthe markers can specifically modulate the activity of a distinct target.In embodiments, the targets can be, for example, at least one of a pairof G_(q)-coupled receptors, a pair of a G_(q)-coupled receptor and aG_(s)-coupled receptor, a pair of a G_(i)-coupled receptor and aG_(s)-coupled receptor, a pair of a G protein-coupled receptor and areceptor-tyrosine kinase, or a pair of a receptor and a cellularprotein. In embodiments, the effect of the ligand candidate on thebiosensor output can be, for example, to modulate of the marker-inducedsignal responses. In embodiments, the modulation can be, for example, achange in signal amplitude, dynamics, kinetics, or a combinationthereof.

In embodiments, the screening method can comprise, for example:

providing a biosensor;

immobilizing a cell line sample expressing at least two differenttargets (i.e., a first target and a second target) onto the biosensorsurface;

incubating the cell line sample with a cocktail solution containing atleast one blocker that inhibits the activity of cellular proteins whichare not the targets but interfere with the activity of the targets;

incubating the cell line sample contacted with the biosensor with aligand candidate for a suitable time;

incubating the ligand candidate-treated cell line with a mixturecontaining at least two activators, each activator selectively activatesone of the different targets;

monitoring the biosensor output during the incubations; and

determining the effect of the ligand candidate on the mixture-inducedbiosensor output.

In embodiments, the blocker(s) and the ligand candidate can be addedseparately or together. If added separately, the blocker(s) is(are)preferably added before the ligand candidate. The blocker can be, forexample, an antagonist for a cellular protein, an inhibitor for acellular protein, an interference RNA (RNAi) for a cellular protein, ananti-sense nucleic acid for a cellular protein, or like entities. Inembodiments, the blocker, such as a solution, can be, for example, addedbefore the ligand candidate. In embodiments, the blocker, such as asolution, can be, for example, added together with the ligand candidate.

In embodiments, the screening method can comprise, for example:

providing a biosensor;

immobilizing a mixed population of cells containing a first cell lineexpressing a first target and a second cell line expressing a secondtarget onto the biosensor surface;

contacting, such as incubating, the immobilized mixed population ofcells on the biosensor with a ligand candidate for a suitable orsufficient time;

incubating the ligand candidate-treated cell line with a mixturecontaining two activators, one activator selectively activates the firsttarget, and the another activates the second target;

monitoring the biosensor output during the incubations; and

determining the effect of the ligand candidate on the mixture-inducedbiosensor output.

In embodiments, the two cell lines can be related each other, forexample, a parental cell line and an engineered cell line using theparental cell. Alternatively, the two cells can be different in origin.

In embodiments, the screening method can comprise, for example:

providing a biosensor;

immobilizing a mixed population of cells containing a first cell lineexpressing a target and a second cell line not expressing the targetonto the biosensor surface;

incubating the cell lines contacted with the biosensor with a ligandcandidate for a suitable or sufficient time;

incubating the ligand candidate-treated cell lines with, for example, asolution containing an activator that selectively activates the target;

monitoring the biosensor output during the incubations; and

determining the effect of the ligand candidate on the activator-inducedbiosensor output.

In embodiments, the two cell lines are preferably related each other,for example, an engineered cell expressing a GPCR, and a parental cellnot expressing the GPCR. The seeding numbers of both cell lines can bepredetermined such that after culture the desired ratio between the twotypes of cells can be, for example, about 1 to about 1. When theresultant mixed population of cells are stimulated with an activator forthe target, the average response obtained will be about 50% compared tothat using only the cells expressing the target. When a ligand candidateis also an activator specific to the target it can provide a comparableresponse as the activator. When a ligand candidate is an activatornon-specific to the target it can provide a different response.

In embodiments, the screening method can comprise, for example:

providing a biosensor;

immobilizing a mixed population of cells containing a first cell lineexpressing a first target and a second cell line sample expressing asecond target onto the biosensor surface;

incubating the cell line sample with a cocktail solution containing atleast one blocker that inhibits the activity of cellular proteins thatare not the targets but interfere with the activity of at least one ofthe targets;

incubating the cell line sample contacted with the biosensor with aligand candidate for a suitable or sufficient time, for example todetect or measure any ligand interaction with the cell, or morespecifically a cell target or receptor;

incubating the ligand candidate-treated cell line with a mixturecontaining at least two activators, each activator selectively activatesone of the different targets; monitoring the biosensor output during theincubations; and

determining the effect of the ligand candidate on the mixture-inducedbiosensor output.

In embodiments, the biosensor can be, for example, an optical biosensor,particularly a resonant waveguide grating biosensor or, for example, anelectrical biosensor, particularly a bio-impedance biosensor. The cellscan be a single cell-type, which expresses two distinct receptors, suchas receptor A and receptor B (FIG. 1, 165, 170). Alternatively, thecells can be two different types of cells, each expressing one receptor,wherein the two types of cells can be mixed together before being placedon the sensor surface. Both receptors can belong to the same type ofreceptors (e.g., G_(q)-coupled receptors), different types of receptors,such as a pair of G_(q) and G_(s)-coupled receptors, a pair of GPCR andreceptor tyrosine kinase, or like receptor combinations. When the tworeceptors lead to similar signaling pathways, the concentrations oftheir corresponding activators can be preferably around their EC₅₀values (i.e., a concentration of activator that binds to and activatesthe receptor, leading to 50% of its maximal response). When the tworeceptors lead to different signaling pathways, the concentrations oftheir corresponding activators can be over a wide range, e.g., fromabout EC₁₀ to about 10×EC₁₀₀, or higher.

In embodiments, the disclosure provides a method that uses a combinationof two different agonists or activators, each activating a differenttarget, for compound screening and profiling. The method can be usefulfor target-specific based screening and profiling, and is particularlywell suited for duplexed or multiplexed receptor or target screening.

In embodiments the present disclosure provides multiplexedtarget-specific compound screening and profiling in a live-cellenvironment using a label-free biosensor, such as a RWG biosensor or anelectric impedance biosensor. The disclosure eliminates the need forcell engineering, or like manipulations, and eliminates the need forhaving the same target classes that require similar or identical cellsignaling pathways, such as Ca²⁺ mobilization. However, engineered cellsor manipulation of cells can be used.

In embodiments, the targets can belong to the same family (e.g.,G_(q)-coupled receptors), or distinct families (e.g., one can be aG_(q)-coupled receptor, another is a G_(s)-coupled receptor;alternatively, one can be a G_(s)-coupled receptor, another can be aG_(i)-coupled receptor; a pair of a G protein-coupled receptor and areceptor tyrosine kinase; a pair comprising a receptor and anintracellular kinase such as protein kinase C; or a pair of anintracellular kinase such as a protein kinase C and an enzyme such asadenylyl cyclase).

In embodiments, the activators or markers can be agonists for receptors,or activators for kinases or enzymes. For a given cell or cell system, apanel of activators, each of which can result in a reliable anddetectable biosensor signal, can be predetermined and selected. Forexample, when an optical biosensor such as RWG biosensor is used, inhuman epidermoid carcinoma A431 cells a panel of activators can beselected from the following group or groups:

An agonist or a partial agonist for endogenous GPCRs can be, forexample, bradykinin for bradykinin B2 receptor, epinephrine for β2adrenergic receptor, adenosine for adenosine A2B receptor, thrombin orSFLLR-amide for protease activated receptor subtype 1, trypsin orSLIGKV-amide for protease activated receptor subtype 2, histamine forhistamine H1 receptor, adenosine triphosphate (ATP) for P2Y receptors,lysophosphatidic acid (LPA) for LPA receptors, see for example, Fang,Y., et al., J. Pharmacol. Tox. Methods, 2007, 55, 314-322.

An agonist for endogenous receptor tyrosine kinase can be, for example,epidermal growth factor (EGF) for EGFR, see for example, Fang, Y., etal., Anal. Chem., 2005, 77, 5720-5725.

An ion channel opener for an endogenous ion channel can be, for example,pinacidil for ATP-sensitive potassium ion channel.

An activator for a cellular enzyme can be, for example, forskolin foradenylate cyclase.

An activator for a cellular kinase can be, for example, 12-deoxyphorbol13-acetate and phorbol 12-myristate 13-acetate for protein kinase C; or8-bromo-cAMP and Sp-cAMPS and dibutyryl-cAMP for protein kinase A.

A disrupting agent can be, for example, cytochalasin D for actinfilament, or nocodozale for microtubules.

An activator for integrin receptor can be, for example, solublefibronectin or its fragments.

A cell membrane disrupting agent can be, for example, saponin to causecell membrane leakage, see for example, Fang, Y., et al., FEBS Lett.,2005, 579, 4175-4180.

An apoptotic inducer can be, for example, Ca²⁻ ionophore A23187 totrigger a Ca²⁺ dependent cell apoptosis.

A kinase inhibitor can be, for example, Y-27632 for Rho kinase.

Since stimulation of the cells examined with each marker leads to aspecific cellular event, a signaling pathway, or signaling networkinteractions, and each signaling pathway may involve distinct sets ofcellular targets, the selected panel of markers will cover many, if notall, of the cellular signaling pathways in the given cell system.

In embodiments the disclosure provides methods for target-specificmultiplexed screening and profiling of compounds using biosensors.Significant attributes of the disclosed method includes, for example:

an applicability to either optical biosensors or electric impedancebiosensors in cell-based assays;

at least a doubling of the assay throughput;

broad applicability to different classes of targets which can be assayedsimultaneously in a single assay; and

the method can be suitable for measuring receptor-receptor interactions.

The receptor-receptor interactions can occur different levels, forexample, dimerization of the two receptors, oligomerization of thereceptors, cross-talk through their down-stream signaling cascades, orlike interactions, and combinations thereof. Conventional cell-basedassays rely on the measurement of a specific cellular event mediatedthrough a target receptor, such as a G protein-coupled receptor. Becauseof that dimension, together with the knowledge that different classes oftarget receptors result in distinct cell signaling in a given cell-type,screening against multiple target receptors largely remains elusive.

Although standard screening campaigns, which typically can involveassaying a single target at a time, have been successful for identifyingpotent drug candidates, very little information about compoundselectivity is generated. Currently, selectivity studies are conducteddownstream in the drug discovery process. However, discarding compoundsat later stages because of adverse binding makes the drug discoveryprocess more expensive and time consuming. Multi-target screens thatexamine the activity of compounds against multiple targets in parallelcould be extremely beneficial in efficiently addressing compoundselectivity at an early stage in the drug discovery process.

Common multiplexed screening methods are typically based on microarraytechnology, which has become a versatile tool for the simultaneousanalysis of many genes and proteins in a single experiment. The use ofprotein microarrays has been extended from profiling protein abundanceto determining, for example, the location, modification, and interactionof proteins with other chemical and biological molecules. Thesedevelopments have created a new paradigm in the drug discovery anddevelopment processes. Examples of microarrays for compound profilingand screening include, for example, air-stable GPCR microarrays, see forexample, Fang, Y., et al., “Membrane protein microarrays”, Journal ofthe American Chemical Society, 2002, 124, 2394-2395; Fang, Y., et al.,“Air-stable G protein-coupled receptor microarrays and ligand bindingcharacteristics”, Analytical Chemistry, 2006, 78, 149-155, cell arraysusing cells cultured on bar-coded CellCard carriers, see for example,Beske, O., et al., “A novel encoded particle technology that enablessimultaneous interrogation of multiple cell types,” Journal ofBiomolecular Screening, 2004, 9, 173-185, or transfected cell clusterarrays using solid state transfection, see for example, Mishina, Y. M.,et al., “Multiplex GPCR assay in reverse transfection cell microarrays,”Journal of Biomolecular Screening, 2004, 9, 196-207. These technologiesgenerally require the engineering or manipulation of cells, such aspurified cell membrane fragments from lysed cells having a targetreceptor over-expressed for GPCR microarrays, or transfections for cellcluster arrays, or distinct types of cells or engineered variants of asingle type of cells for CellCard technology.

Duplexed functional assays have also been developed based on Ca²⁺ fluxmeasurements, for example, by mixing two stably transfected cellpopulations in the right proportions, where each population expresses atarget. If there is a “hit” that can activate one receptor, it canresult in 50% of the fluorescence signal using, for example, a FLIPR(fluorometric imaging plate reader) system. Additionally, a “hit” couldresult in 100% of the signal if another hit has cross-activity and canactivate the two receptors. Such a screen would double the potentialhits generated using a single-target screen. However, additional screenswould be needed using a single-target screen to de-convolute the hits.Such a duplexed assay is only limited to two G_(q)-coupled receptors inwhich both mediate signaling through G_(q) and subsequent Ca²⁺mobilization.

In embodiments the disclosure provides a label-free screening methodcomprising:

providing an biosensor having a mixed population of cells containing twotypes of cells co-immobilized on a surface of the biosensor;

contacting the immobilized cells with a ligand candidate; and

determining the ligand candidate-induced biosensor output.

The two types of cells can be, for example, a parental cell and anengineered cell expressing a target; two engineered cells eachexpressing a target; or two native cells.

In embodiments the disclosure provides a label-free screening methodcomprising:

providing a biosensor having a mixed population of cells containing afirst type of cells expressing a first target and a second type of cellsexpressing a second target, both cell types are co-immobilized on asurface of the biosensor;

contacting the immobilized cells with a ligand candidate;

contacting the ligand candidate-treated cells with a mixture containingtwo markers, each marker specifically modulating the activity of atarget; and

determining the effect of the ligand candidate on the markermixture-induced biosensor output.

1. Label-Free Biosensor-Based Cell Assays

Label-free cell-based assays generally employ a biosensor to monitorligand-induced responses in living cells. A biosensor typically utilizesa transducer such as an optical, electrical, calorimetric, acoustic,magnetic, or like transducer, to convert a molecular recognition eventor a ligand-induced change in cells contacted with the biosensor into aquantifiable signal. These label-free biosensors can be used formolecular interaction analysis, which involves characterizing howmolecular complexes form and disassociate over time, or for cellularresponse, which involves characterizing how cells respond tostimulation. FIG. 1 highlights two types of biosensors that arecurrently used as the basis for label-free cell-based assays, resonantwaveguide grating (RWG) biosensors and electrical biosensors, and how touse biosensor-based cell assays for dual target-based screening andprofiling of compounds.

RWG biosensors and systems—An RWG biosensor can include, for example, asubstrate (e.g., glass), a waveguide thin film with an embedded gratingstructure, and a cell layer (FIG. 1 a). The RWG biosensor utilizes theresonant coupling of light into a waveguide by means of a diffractiongrating, leading to total internal reflection at the solution-surfaceinterface, which in turn creates an electromagnetic field at theinterface. This electromagnetic field is evanescent in nature, meaningthat it decays exponentially from the sensor surface; the distance atwhich it decays to 1/e of its initial value is known as the penetrationdepth and is a function of the design of a particular RWG biosensor, butis typically on the order of about 200 nm. This type of biosensorexploits such evanescent waves to characterize ligand-inducedalterations of a cell layer at or near the sensor surface.

RWG instruments can be subdivided into systems based on angle-shift orwavelength-shift measurements. In a wavelength-shift measurement,polarized light covering a range of incident wavelengths with a constantangle is used to illuminate the waveguide; light at specific wavelengthsis coupled into and propagates along the waveguide. Alternatively, inangle-shift instruments, the sensor is illuminated with monochromaticlight and the angle at which the light is resonantly coupled ismeasured. The resonance conditions are influenced by the cell layer(e.g., cell confluency, adhesion and status), which is in direct contactwith the surface of the biosensor. When a ligand or an analyte interactswith a cellular target (e.g., a GPCR, a kinase) in living cells, anychange in local refractive index within the cell layer can be detectedas a shift in resonant angle (or wavelength).

The Corning® Epic® system uses RWG biosensors for label-free biochemicalor cell-based assays (Corning Inc., Corning, N.Y.). The Epic® Systemconsists of an RWG plate reader and SBS (Society for BiomolecularScreening) standard microtiter plates. The detector system in the platereader exploits integrated fiber optics to measure the shift inwavelength of the incident light, as a result of ligand-induced changesin the cells. A series of illumination-detection heads are arranged in alinear fashion, so that reflection spectra are collected simultaneouslyfrom each well within a column of a 384-well microplate. The whole plateis scanned so that each sensor can be addressed multiple times, and eachcolumn is addressed in sequence. The wavelengths of the incident lightare collected and used for analysis. A temperature-controlling unit canbe included in the instrument to minimize spurious shifts in theincident wavelength due to the temperature fluctuations.

Electrical biosensors and systems—Electrical biosensors consist of asubstrate (e.g., plastic), an electrode, and a cell layer (FIG. 1 b). Inthis electrical detection method, cells are cultured on small goldelectrodes arrayed onto a substrate, and the system's electricalimpedance is followed with time. The impedance is a measure of changesin the electrical conductivity of the cell layer. Typically, a smallconstant voltage at a fixed frequency or varied frequencies is appliedto the electrode or electrode array, and the electrical current throughthe circuit is monitored over time. The ligand-induced change inelectrical current provides a measure of cell response. Impedancemeasurement for whole cell sensing was first realized in 1984. Sincethen, impedance-based measurements have been applied to study a widerange of cellular events, including cell adhesion and spreading, cellmicromotion, cell morphological changes, and cell death. Classicalimpedance systems suffer from high assay variability due to use of asmall detection electrode and a large reference electrode. To overcomethis variability, the latest generation of systems, such as the CellKeysystem (MDS Sciex, South San Francisco, Calif.) and RT-CES (ACEABiosciences Inc., San Diego, Calif.), utilize an integrated circuithaving a microelectrode array.

The CellKey system consists of an environmentally controlled impedancemeasurement system, a 96-well electrode-embedded microtiter plate, anonboard 96-well fluidics, and custom acquisition and analysis software.The cells are seeded in the culture wells; each well has an integratedelectrode array. The system operates using a small-amplitude alternatingvoltage at 24 frequencies, from 1 KHz to 10 MHz. The resultant currentis measured at an update rate of 2 sec. The system is thermallyregulated and experiments can be conducted, e.g., between 28° C. and 37°C. A 96-well head fluid delivery device handles fluid additions andexchanges onboard.

The RT-CES system can include four main components: electronicmicrotiter plates (E-Plate™), E-Plate station, electronic analyzer, anda monitoring system for data acquisition and display. The electronicanalyser sends and receives the electronic signals. The E-Plate stationis placed inside a tissue culture incubator. The E-Plate station comesin three throughput varieties: a 16× station for running six 16-wellE-Plates at a time, a single 96-well E-Plate station, and theMult-E-Plate™ station, which can accommodate up to six 96-well E-Platesat a time. The cells are seeded in E-Plates, which are integrated withmicroelectronic sensor arrays. The system operates at a low-voltage(less than 20 mV) AC signal at multiple frequencies.

Optical signals of GPCR activation with RWG biosensor—Cells are dynamicobjects with relatively large dimensions, e.g., typically tens ofmicrons. RWG biosensors enable detection of ligand-induced changeswithin the bottom portion of cells, determined by the penetration depthof the evanescent wave. Furthermore, the spatial resolution of anoptical biosensor is determined by the spot size (about 100 microns) ofthe incident light source. Thus, a highly confluent cell layer isgenerally used in order to achieve optimal assay results; and the sensorconfiguration can be viewed as a three-layer waveguide composite,including, for example, a substrate, waveguide thin film, and a celllayer. Following a 3-layer waveguide biosensor theory in combinationwith cellular biophysics, we found that for whole-cell sensing, aligand-induced change in effective refractive index, the detected signalΔN, is governed by equation (1):

$\begin{matrix}{{\Delta \; N} = {{{S(N)}\Delta \; n_{C}} = {{S(N)}\alpha \; d{\sum\limits_{i}{\Delta \; {C_{i}\left\lbrack {^{\frac{- z_{l}}{\Delta \; Z_{C}}} - ^{\frac{- z_{l + 1}}{\Delta \; Z_{C}}}} \right\rbrack}}}}}} & (1)\end{matrix}$

where S(C) is the system sensitivity to the cell layer, and Δn_(c) isthe ligand-induced change in local refractive index of the cell layersensed by the biosensor. ΔZ_(c) is the penetration depth into the celllayer, α is the specific refractive index increment (about 0.18/mL/g forproteins), z_(i) is the distance where the mass redistribution occurs,and d is an imaginary thickness of a slice within the cell layer. Herethe cell layer is divided into an equally-spaced slice in the verticaldirection. We assumed that the detected signal is, to a first order,directly proportional to the change in refractive index of the bottomportion of cell layer Δn_(c). The Δn_(c) in turn is directlyproportional to changes in local concentration of cellular targets ormolecular assemblies within the sensing volume, given the refractiveindex of a given volume within cells is largely determined by theconcentration of biomolecules, mainly proteins. A weighted factorexp(−z_(i)/ΔZ_(c)) is taken into account for a change in local proteinconcentration that occurs, considering the exponentially decaying natureof the evanescent wave. Thus, the detected signal is the sum of massredistribution occurring at distinct distances away from the sensorsurface, each with unequal contribution to the overall response. Eq. (1)suggests that the detected signal with an RWG biosensor is sensitiveprimarily to the vertical mass redistribution, as a result of a changein local protein concentration. The detected signal is often referred toas a dynamic mass redistribution (DMR) signal.

GPCR activation leads to a series of spatial and temporal events,including, for example, ligand binding, receptor activation, proteinrecruitment, receptor internalization and recycling, second messengeralternation, cytoskeletal remodeling, gene expression, and cell adhesionchanges. Each cellular event has its own characteristics regarding itskinetics, duration, amplitude, and mass movement. Thus it is reasonableto assume that these cellular events may contribute differently to theoverall DMR signal, depending on where they occur. Using a panel ofagonists targeting a variety of GPCRs, we have identified three classesof DMR signals in human epidermoid carcinoma A431 cells, which reflectthe signaling pathways mediated. Since each is correlated with theactivation of a class of GPCRs depending on the G protein with which thereceptor is coupled, the DMR signals obtained were named G_(q)-, G_(s)-and G_(i)-DMR signals, respectively. Each class of DMR signals exhibitsdistinct kinetic and dynamic characteristics, reflecting the uniquesignaling integration mediated through different classes of GPCRs. Theunique characteristics of the DMR signals can be used to identify theG-protein coupling mechanism of orphan GPCRs.

Bioimpedance signals of GPCR activation—In a typical impedance-basedcell assay, cells are brought into contact with a gold electrode arrayedon the bottom of culture wells. The total impedance of the sensor systemis determined primarily by the ion environment surrounding thebiosensor. Under application of an electrical field, the ions undergofield-directed movement and concentration gradient-driven diffusion. Forwhole cell sensing, the total electrical impedance has four components:the resistance of the electrolyte solution; the impedance of the cell;the impedance at the electrode/solution interface; and the impedance atthe electrode/cell interface. In addition, the impedance of a cellcomprises two components: the resistance; and the reactance. Theconductive characteristics of cellular ionic strength provide theresistive component, whereas the cell membranes, acting as imperfectcapacitors, contribute a frequency-dependent reactive component. Thus,the total impedance is a function of many factors, including, forexample, cell viability, cell confluency, cell numbers, cell morphology,degree of cell adhesion, ionic environment, the water content within thecells, the detection frequency, and like considerations.

In the RT-CES system, a percentage of this small voltage applied iscoupled into the cell interior. Such signals applied to cells arebelieved to be much smaller than the resting membrane potential of atypical mammalian cell and thus present minimal or no disturbance tocell function. The RT-CES system measures these changes in impedance anddisplays it as a parameter called the cell index. The cell index iscalculated according to the equation (2):

$\begin{matrix}{{CI} = {\max\limits_{{i = 1},\ldots \mspace{14mu},N}\left( {\frac{R_{cell}\left( f_{i} \right)}{R_{0}\left( f_{i} \right)} - 1} \right)}} & (2)\end{matrix}$

where N is the number of frequency points at which the impedance ismeasured (e.g., N=3 for 10 kHz, 25 kHz, and 50 kHz), and R₀(f) andR_(cell)(f) are the frequency electrode resistance without cells or withcells present in the wells, respectively.

In the CellKey system, a change in sensor system's impedance isattributed to a change in complex impedance (delta Z or dZ) of a celllayer that occurs in response to receptor stimulation. At lowfrequencies, the small voltage applied induces extracellular currents(iec) that pass around individual cells in the layer. However, theconduction currents through cell membrane due to ion channels may alsobe important at low measurement frequencies. At high frequencies, theyinduce transcellular currents (itc) that penetrate the cellular membrane(FIG. 1 b). The ratio of the applied voltage to the measured current foreach well is its impedance (Z) as described by Ohm's law.

When cells are exposed to a stimulus, such as a receptor ligand, signaltransduction events are activated that lead to complex cellular eventssuch as modulation of the actin cytoskeleton that cause changes in celladherence, cell shape and volume, and cell-to-cell interaction. Thesecellular changes individually or collectively affect the flow ofextracellular and transcellular current, and therefore, affect themagnitude and characteristics of the measured impedance. For example, aCellKey system was used to identify the impedance responses of cellsmediated through the activation of distinct classes of GPCRs. Resultsshowed that there are three types of impedance signals mediated throughthe activation of three classes of GPCRs, depending on the G protein towhich the receptor is coupled. Similar profiles were also recorded usingthe RT-CES system. Although not limited by theory it is believed thatthese impedance signals are due to the different effects on the actincytoskeleton that affect the cellular parameters measured by impedance,in response to the activation of different classes of GPCRs. It has beenshown that activation of G_(q) and G_(i) GPCRs leads to increased actinpolymerization, while stimulation of G_(s) GPCRs leads to actindepolymerization.

Both optical and electrical biosensors are applicable to many distinctclasses of targets, including GPCRs, receptor tyrosine kinases, kinases,enzymes, or other cellular targets.

2. Duplexed Target-Specific Screening Using Biosensor-Based Cell Assays

The biosensor-based cell assays are capable of multiplexing. Theactivation of a same class of targets (e.g., G_(q)-coupled receptors) ina given cell line leads to almost identical optical signatures,suggesting that multiple targets within the same family can be assayedat the same time. For example, A431 cells endogenously expressbradykinin B₂ receptor, P2Y receptors, and protease activated receptors(PARs). Upon stimulation with bradykinin, ATP, or thrombin, quiescentA431 cells respond with similar G_(q)-type optical signatures. Thequiescent state is obtained through continuous culturing using aserum-free medium for about 20 hours. Fluo-3 assays show that theactivation of all three receptors mediates G_(q)-signaling. Theseobservations suggest that biosensor-based assays can be used to screencompounds or hits that can activate same classes of receptors, which areexpressed within the same cells.

G protein-coupled receptors (GPCRs) are the richest class of drugtargets in the human genome and remain a popular target for thepharmaceutical industry. About 30 known GPCRs are the targets for about40% of all currently marketed drugs and many other functionallyuncharacterized GPCRs are potentially druggable targets and represent anuntapped resource in drug discovery. Efforts to bring new GPCR drugs tothe market has prompted a revolution in assay methods, particularlyfunctional cellular assays. However, current assays are mostlypathway-biased and only measure “points of contact” in GPCR signalingcascades. Given the recent realization of the complexity of GPCRsignaling and of the ligand-directed functional selectivity, thesepathway-biased assays tend to result in false negatives. Furthermore,many conventional assays typically assay a single target at a timebecause of their limited capacity for multiplexing and theirpathway-biased nature. Multi-target screens that can examine theactivity of compounds against multiple targets simultaneously arelogically suited to address compound selectivity.

Label-free optical biosensors including surface plasmon resonance (SPR),resonant waveguide grating (RWG), and plasmon-waveguide resonance (PWR)are routinely used for biomolecular interaction analysis. Recently,label-free optical biosensors were applied for whole cell sensing, andthese biosensors are capable of monitoring endogenous receptoractivation, leading to high-information and physiologically relevantmeasures of a receptor-ligand pair (see Fang, Y. et al. “Resonantwaveguide grating biosensor for living cell sensing”, Biophys. J., 2006,91, 1925-1940). These assays do not require prior knowledge of cellsignaling, and are pathway-unbiased (see Fang, Y. et al., “Non-invasiveoptical biosensor for assaying endogenous G protein-coupled receptors inadherent cells”, J. Pharmacol. Toxicol. Methods, 2007, 55, 314-322). Theoptical responses recorded are pathway-sensitive, and reflect thecomplexity of receptor signaling (see Fang, Y., et al., “Opticalbiosensor provides insights for bradykinin B2 receptor signaling in A431cells”, FEBS Lett., 2005, 579, 6365-6374).

Referring to the figures, FIG. 1 show principles of biosensor-based cellassays and duplexed target-specific screening. FIG. 1 a shows an RWGbiosensor for monitoring ligand-induced dynamic mass redistribution(DMR) in living cells (100). Cells can be directly cultured onto thesurface of the biosensor, or brought to contact with the sensor surface.In embodiments, the biosensor can include, for example, a glasssubstrate (105), a waveguide (110) thin film within which a gratingstructure is embedded, a light source (112), and means to detect andprocess the resulting refracted light (113). Only the massredistribution within the detection zone (115) and the bottom portion ofcells (120) is directly measured. FIG. 1 b is an electric biosensor formonitoring the ionic environment surrounding the biosensor and the cells(150). Cells can be cultured on the surface of a biosensor having, forexample, arrayed gold microelectrode(s) (155) on a substrate (160). Bothextracellular (161) and transcellular (162) current flows can bemeasured, while a low AC voltage at variable frequencies (e.g., electricpulse (180)) is applied to the cell. In FIGS. 1 a and 1 b the squares(▪) represent a ligand (163) for Receptor A (165), while circles (∘)represent a ligand (168) for Receptor B (170). Both receptors A and Bare expressed in the same cell.

Experimental Procedures Materials

The LOPAC was purchased from Sigma Chemical Co. (St. Louis, Mo.).S(−)epinephrine, dopamine, norepinephrine, and histamine were obtainedfrom Tocris (St. Louis, Mo.). (±)-brompheniramine maleate,(±)chlorpheniramine maleate, clemizole hydrochloride, clemastinefumarate, diphenhydramine hydrochloride, or triprolidine hydrochloride,SKF91488 dimaleate, ranitidine hydrochloride, catechol and thioperamidemaleate were purchased from Sigma (St. Louis, Mo.). SFFLR-amide wasobtained through Bachem (King of Prussia, Pa.). Cell culture compatibleEpic® 384-well RWG (resonant waveguide grating) biosensor microplateswere obtained from Corning Inc (Corning, N.Y.).

Cell Culture

Human epidermoid carcinoma A431 cells (American Type Cell Culture) weregrown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%fetal bovine serum (FBS), 4.5 g/liter glucose, 2 mM glutamine, andantibiotics. About 1.8×10⁴ cells at passage 3 to 15 suspended in 50 μLthe medium containing 10% FBS were placed in each well of a 384-wellmicroplate, and were cultured at 37° C. under air/5% CO₂ for about 1day, followed by about 20 hr starvation through continuously culture inthe serum-free DMEM.

Optical Biosensor System and Cell Assays

Corning® Epic® wavelength interrogation system was used. This systemconsists of a temperature-control unit, an optical detection unit, andan on-board liquid handling unit with robotics. The detection unit iscentered on integrated fiber optics, and enables kinetic measures ofcellular responses with a time interval of about 7 or about 15 sec.

The RWG biosensor exploits its evanescent wave, created by the totalinternal reflection of light at a solution-surface interface, to measureligand-induced dynamic mass redistribution (DMR) signals in cells. Theevanescent wave extends into the cells and exponentially decays overdistance, leading to a characteristic sensing volume of about 150 nm,implying that any optical response mediated through the receptoractivation only represents an average over the portion of the cell thatthe evanescent wave is sampling. Such sampling with the biosensor issufficient to differentiate the signaling of distinct classes of GPCRsin living cells, and offers a simplified representation of GPCRsignaling.

Like SPR, the RWG biosensor is sensitive to refractive index—anintrinsic property of biomolecules. Since the refractive index of agiven volume within a cell is largely determined by the concentrationsof bio-molecules such as proteins, we found, based on a three-layerwaveguide grating theory, that a ligand-induced optical response islargely associated with dynamic mass redistribution. The relocation ofcellular targets towards the sensor surface (e.g., relocation ofintracellular targets to the activated receptors at the basal membranesurface) makes a positive contribution to the DMR (P-DMR); conversely,the movement of cellular targets away from the sensor surface (e.g.,receptor internalization) is a negative contributor to the DMR (N-DMR).The aggregation of these events determines the kinetics and amplitudesof a ligand-induced DMR. However, recent studies, using PWR technologyand in vitro reconstituted GPCRs immobilized onto the sensor surface,showed that a ligand-induced optical response of the receptor-lipidmembrane system consists of two components—changes in mass density andchanges in structure. Since the RWG biosensor used here is unable todifferentiate the contributions of these components, ligand-inducedchanges in organization of biomolecules in living cells may alsocontribute to the overall response measured.

For biosensor cellular assays, a 2 min baseline was first established.Compound solutions were then transferred into the sensor plate havingcells maintained in Hanks balanced salt solution (20 mM Hepes, pH 7.1),and the cell responses were recorded continuously for one hour.Afterwards, a second baseline (about 2 min) was established, thecocktail solution containing 2 nM epinephrine and 500 nM histamine wasintroduced to each well. The co-stimulation response was continuouslymonitored for an additional one hour. All studies were carried out atcontrolled temperature (28° C.) and with three replicates for eachmeasurement, unless specifically mentioned. The assay coefficient ofvariation was found to be <10%. All data analysis was carried out usingeither Microsoft Excel, or Prism software (Graph Pad).

EXAMPLES

The following examples serve to more fully describe the manner of usingthe disclosure, as well as to further illustrate and demonstratespecific examples of best modes contemplated for carrying out variousaspects of the disclosure. These examples do not limit the scope of thedisclosure, but rather are presented for illustrative purposes.

Example 1

Optical responses of quiescent A431 cells upon c-stimulation with bothSFFLR-amide and epinephrine, or both bradykinin and epinephrine Thenotion of compartmentalization in which unique changes in secondmessenger levels occur in both time and space was established with theadvent of high resolution single-cell imaging systems in combinationwith fluorescent probes. It is widely believed that GPCR signalingproceeds through a series of highly regulated spatial and temporalevents besides the production and regulations of second messengers suchas Ca²⁺ and cAMP. However, such “tunneling” or “channeling” of theseries of signaling events through a receptor has not been previouslydemonstrated or established. The label-free optical biosensors enablethe measurement of an integrated cellular response relating toligand-induced dynamic mass redistribution in cells within the detectionzone of the biosensor, offering a novel readout for receptor biology andligand pharmacology. Here the biosensor cellular assays were used toinvestigate the optical response of quiescent A431 cells in response toco-stimulation with SFLLR-amide and epinephrine. SFFLR-amide mediatedsignaling through endogenous G_(q)-coupled protease activated receptors(see Fang, Y. and Ferrie, A. M. “Optical biosensor differentiatessignaling of endogenous PAR1 and PAR2 in A431 cells,” BMC Cell Biol.,2007, 8, 24), whereas epinephrine mediated signaling is throughendogenous G_(s)-coupled β2-adrenergic receptor (β2AR) (see Fang, Y. etal., “Non-invasive optical biosensor for assaying endogenous Gprotein-coupled receptors in adherent cells,” Journal of Pharmacological& Toxicological Methods, 2007, 55, 314-322).

FIG. 2 provide examples of RWG biosensor-based cell assays against twoclasses of G protein-coupled receptors. FIG. 2 a shows G_(q)-coupledreceptor (protease activated receptor subtype 1, PAR₁) (210) andG_(s)-coupled receptor (β2 adrenergic receptor, β2AR) (220) in A431cells. A431 cells endogenously express both PAR₁ and β2AR. SFLLR-amide(215) is a PAR₁-specific agonist, while epinephrine (225) is aβ2AR-specific agonist. FIG. 2 b shows that stimulation of A431 cellswith, for example, 1 micromolar SFLLR-amide leads to a G_(q)-type DMRsignal. FIG. 2 c shows that stimulation of A431 cells with, for example,2 nanomolar epinephrine leads to a G_(s)-type DMR signal. This isconsistent with the known signaling pathways mediated through eachreceptor individually, i.e., PAR₁ activation leads to a G_(q) pathway,while β2AR activation leads to a G_(s) pathway. FIG. 2 d shows thatco-stimulation of A431 cells with SFLLR-amide (1 micromolar) andepinephrine (2 nanomolar) leads to an optical signature that appears tobe the sum of the optical signatures induced by SFLLR-amide andepinephrine individually. In FIGS. 2 b, 2 c and 2 d, the error bar ateach time point was also included. One possible explanation is thatdistinct classes of GPCRs can mediate signaling through distinct routes(e.g., “channeling” or “tunneling”), and the cells can respondsynergistically to co-stimulation with two agonists targeting twodifferent receptors.

Similar results were observed for co-stimulation of A431 cells withbradykinin and epinephrine. FIG. 3 provides an example of RWGbiosensor-based cell assays against two classes of G protein-coupledreceptors: G_(q)-coupled receptor (bradykinin B2 receptor,) andG_(s)-coupled receptor (β2 adrenergic receptor, β2AR) in A431 cells.A431 cells endogenously express both B2 receptor and β2AR. Bradykinin isa B2-specific agonist, while epinephrine is a β2AR-specific agonist.FIG. 3 a shows that stimulation of A431 cells with 16 nanomolarbradykinin leads to a G_(q)-type DMR signal. FIG. 3 b shows thatstimulation of A431 cells with 2 nanomolar epinephrine leads to aG_(s)-type DMR signal. This is consistent with the known signalingpathways mediated through each receptor, i.e., B2 activation leadsprimarily to a G_(q) pathway, whereas β2AR activation leads to a G_(s)pathway. FIG. 3 c shows that co-stimulation of A431 cells withbradykinin (2 nanomolar) and epinephrine (2 nanomolar) leads to anoptical signature that closely resembles the sum of the opticalsignatures induced by bradykinin and epinephrine individually. In FIGS.3 a, 3 b and 3 c, the error bar at each time point was also included toshow that the kinetic response is highly reproducible.

Example 2

Optical responses of quiescent A431 cells upon co-stimulation withepinephrine and histamine A431 endogenously expresses large numbers ofthe β₂AR, but neither β₁ nor β₃-ARs. Epinephrine mediated a biphasicG_(s)-type DMR response in quiescent A431 cells (410) (FIG. 4). The DMRsignal consists of a small decreased signal (i.e., N-DMR) with a shortduration, followed by an increased signal (i.e., P-DMR) to an elevatedlevel. The epinephrine response was dose-dependent and saturable (520),leading to an apparent EC₅₀ of 0.08±0.03 nM and a Hill slope of 0.95(n=10) (FIG. 5 a and FIG. 5 c). In FIG. 5 a, cells were stimulated withepinephrine at different doses (501: 0.01 nanomolar (nM); 502: 0.02 nM;503: 0.04 nM; 504: 0.08 nM; 505: 0.16 nM; 506: 0.64 nM; 507: 2.56 nM;508: 10.25 nM), whose real time kinetic responses were recorded. Theβ-blocker propranolol dose-dependently attenuated the epinephrineresponse (data not shown), suggesting that the epinephrine response isβ₂AR-specific.

A431 also endogenously expresses histamine receptor subtype 1 (H₁R). Thestimulation of quiescent A431 cells with histamine led to adose-dependent and saturable G_(q)-type optical signal, which consistsof an initial P-DMR and a subsequent N-DMR (420) (FIG. 4). The histamineresponses were also saturable (FIG. 5 b). In FIG. 5 b, cells werestimulated with histamine at different doses: 511: 2.7 nM; 512: 43.5 nM;513: 174 nM; 514: 696 nM; 515: 2,784 nM; 516: 11,138 nM; 517: 445,506nM. The saturation curve (530), as plotted as the P-DMR amplitude as afunction of histamine concentration, appeared to fit well with sigmoidalnon-linear regression with variable slope, leading to an apparent EC₅₀of 687±34 nM (n=6) and a Hill coefficient of 1.87 (FIG. 5 c). The verysteep activation curve observed suggests that there is positivecooperativity of the receptor through unknown mechanism(s). Thepretreatment of A431 with H₁ specific antagonists (±)-brompheniramine,(±)chlorpheniramine, clemizole, clemastine, diphenhydramine, ortriprolidine, each at 1 μM, completely inhibited the DMR signal inducedby 1 μM histamine. Conversely, the H₂ antagonists SKF91488 andranitidine at 1 μM did not have obvious impact on the histamineresponse, nor did H₃-specific antagonist thioperamide. These resultssuggest that the histamine response is largely H₁R-specific.

Compartmentalized signaling in which unique changes in second messengerlevels occur in both time and space has been established and is known tobe central in GPCR signaling. Since the DMR signal is a global measureof receptor signaling at least in terms of mass redistribution withinthe sensing volume of the biosensor, the co-activation of two receptorscoupled to distinct classes of G proteins could lead to a DMR signalthat is largely a sum of the two DMR signals mediated through theactivation of each receptor. To test this hypothesis, quiescent A431cells were stimulated with epinephrine and histamine individually ortogether. Results showed that indeed stimulation of quiescent A431 cellswith 1 μM histamine and 2 nM epinephrine led to a unique DMR signal(430) that closely resembles the sum (440) of the two DMR signalsobtained individually (FIG. 4). Because of its simply additive nature,the early P-DMR event is referred to the histamine response (450), andthe retarded P-DMR event is the epinephrine response (460). Togetherwith examples shown above, these results represents the first evidencethat distinct classes of GPCRs can mediate signaling mostlyindependently, strongly suggesting that spatial and temporalcompartmentalization of signaling cascades is central to GPCR signaling.Most of cellular events downstream of a receptor signaling occur as“tunneling” or “channeling”.

Interestingly, the co-stimulation DMR signal is not a simple addition ofthe two individual DMR signals—its initial P-DMR was largely identicalto the calculated signal, whereas its second decaying phase exhibitedfaster kinetics and bigger amplitude than the calculated signal.Although not bound by theory this may be due to the crosstalk of thehistamine mediated signaling with the epinephrine mediated signaling. Itis known that the activation of G_(q)-coupled receptors leads to theregulation of adenylate cyclases in cAMP microdomains. To test thispossibility, a desensitization assay was used. Here A431 cells werefirst pre-treated with either epinephrine or histamine at differentdoses, followed by co-stimulation with the cocktail solution containing2 nM epinephrine and 1 μM histamine. Results showed that epinephrinedose-dependently attenuated both the histamine response and theepinephrine response (FIG. 6 a), with almost identical IC₅₀ (0.66±0.20nM, and 0.38±0.09 nM (n=4), respectively) (FIG. 6 b). In FIG. 6 a, thecells were pretreated with epinephrine at different doses (601: 0.01 nM;602: 0.03 nM; 603: 0.13 nM; 604: 0.50 nM; 605: 2 nM; 606: 8 nM; 607: 32nM; 608: 256 nM), followed by co-stimulation with 1,000 nM histamine and2 nM epinephrine. Only real time co-stimulation-induced DMR signal wererecorded and shown in FIG. 6 a. In FIG. 6 b, both the epinephrineresponse (630) and the histamine response (620) were plotted as thefunction of epinephrine concentrations used for the cell pretreatment.Epinephrine at high doses completely inhibited the later epinephrineresponse, but only partially attenuated the early histamine response.Conversely, the pretreatment of cells with histamine completelyinhibited the histamine response with an apparent IC₅₀ of 1.6±0.4 μM anda Hill slope of 1.4 (n=4), but slightly attenuated the epinephrineresponse (FIG. 6 c and FIG. 6 d). In FIG. 6 c, the cells were pretreatedwith histamine at different doses: 611: 2.7 nM; 612: 10.9 nM; 613: 43.5nM; 614: 174 nM; 615: 696 nM; 616: 2,784 nM; 617: 11,138 nM; 618: 44,550nM), followed by co-stimulation with 1,000 nM histamine and 2 nMepinephrine. Only real time co-stimulation-induced DMR signal wererecorded and shown in FIG. 6 c. In FIG. 6 d, both the epinephrineresponse (650) and the histamine response (640) were plotted as thefunction of epinephrine concentrations used for the cell pretreatment.These results suggest that the G_(q)-coupled receptor H₁R indeedcross-talks with G_(s)-coupled receptor β₂AR, possibly through cAMP-PKApathway. Nonetheless, these results demonstrate a dual receptor-specificscreening method using the label-free biosensor cellular assays.

Example 3

Optical biosensor cellular assays for agonist screening using LOPAClibrary acting on A431 cells—The Sigma-Aldrich LOPAC 1280™ library ofcompounds includes 1,280 bioactive small organic molecules against allmajor target classes including several GPCRs, and was chosen to validatescreening using the biosensor cellular assays. Since a ligand-inducedDMR signal is an integrated response and many ligands often exhibitcross-activity to more than one receptor in the cells, the librarymembers were diluted in 1×HBSS containing 0.1% DMSO to achieve 1 microMfinal concentration for screening to minimize the off-target effect.Furthermore, a ligand-induced DMR is a real time kinetic responsecontaining many useful parameters (e.g., phases, amplitudes andkinetics) for analyzing ligand pharmacology. The LOPAC library was usedto screen agonists for endogenous receptors in A431 cells using realtime kinetic measurements. FIG. 7 summarizes some representative classesof DMR signals that were obtained with the library using the method.Histamine led to a biphasic G_(q)-type DMR signal (FIG. 7 a). The β2ARfull agonists (±)isoproterenol and R(−)isoproterenol led to a typicalG_(s)-type DMR signal (FIG. 7 b and FIG. 7 d, respectively). The twoβ₂AR partial agonists (±)CGP12177 and S(−)pindolol led to a G_(s)-likeDMR which only consists of the prolonged P-DMR event without the initialN-DMR event (FIG. 7 c and FIG. 7 e). Ruthernium red, an inhibitor ofmitochondrial Ca²⁺ uniporter and VR1 vanilloid receptor-coupled ionchannel, also led to a G_(s)-like DMR signal but with much largeramplitude (FIG. 7 f). Like many compounds in the library, the potent H₁antagonist triprolidine did not lead to any significant DMR signal(referred to as a net zero-DMR) (data not shown).

Based on the kinetic profiles of both the epinephrine response and thehistamine response, four types of end-point measurements were chosen todetermine the response of compounds in the library. First, the responsein terms of wavelength shift between before and 2 min after stimulationwas calculated. Results showed that using this end-point measurement,only one hit, histamine, was identified from the library (data notshown). Since the initial N-DMR event in the epinephrine was relativelysmall, such end-point measurement appeared to be insufficiently robustto identify G_(s)-coupled receptor agonists. Conversely, since theinitial P-DMR event in the histamine response was quite large, suchend-point measurement was well-suited for identifying agonists forG_(q)-coupled receptors.

Second, the responses in terms of wavelength shift between before and 50min after stimulation were calculated. Results showed that thisend-point measurement was well-suited for identifying agonists forG_(s)-coupled receptors (data not shown). However, it fails to identifyagonists for G_(q)-coupled receptors, since the G_(q)-type DMR consistsof an initial rapid P-DMR and a subsequent slow N-DMR that almost decaysback to the initial baseline (FIG. 4 and FIG. 5 b).

Third, the responses in terms of wavelength shift between 2 min and 50min after stimulation were calculated. The difference in resonantwavelength between the two time points was plotted as a function ofcompounds (FIG. 8). Results showed that such two time point measurementis sufficient and robust to identify agonists in the library for bothG_(q)-coupled receptors and G_(s)-coupled receptors. Similar hits wereselected using a three point measurement, i.e., 2 min before and 50 minafter stimulation (data not shown). Thus, the responses measured withthe wavelength shift from 2 min to 50 min after stimulation were usedfor selecting hits for both H1R and β₂AR. An agonist for H1R would leadto a large negative response, whereas a β₂AR agonist would lead to apositive response.

As shown in FIG. 8, there was only one hit for G_(q)-coupled H1R.Histamine led to a large negative response. In the LOPAC, histamine isthe only broad spectrum histamine receptor agonist, although there aretwo H3R specific agonists R(−)-α-methyl-histamine and imetit. The twoH3R agonists at 1 μM did not lead to any obvious DMR signal. Besidehistamine receptor agonists, P2Y agonists of 1 μM in the library alsodid not lead to any apparent or specific DMR signal, although the A431endogenously expresses G_(q)-coupled P2Y receptors. Although not limitedby theory this was probably due to the low potency of these agonists toactivate P2Y receptors.

Conversely, there were many hits that lead to positive responses havingdistinct amplitudes. Based on the epinephrine positive controls whichlead to a response of 225±18 pm (n=64), hits that led to a response from170 to 280 pm were considered as full or strong partial agonists.Results showed that 63 hits acted as full or strong partial agonists—12adenosine receptor agonists, 27 adrenoceptor agonists, and 7 dopaminereceptor agonists. In addition, hits that lead to a response from 40 to170 were considered as partial or weak partial agonists. Results showedthat 51 hits fell into this category, including 6 adenosine agonists, 6adrenoceptor agonists, and 5 dopamine agonists. This high positive hitrate reflects the fact that A431 also endogenously expresses adenosinereceptors, whose activation also leads to an epinephrine-like DMRresponse. Furthermore, many dopamine agonists are known to activate theβ2AR. Except for two low potency beta-adrenoceptor agonists,(±)-ephedrine and amiodarone, all other beta-adrenoceptor agonists inthe library were correctly identified. Such low false negative ratedemonstrates in-part the viability of the disclosed biosensor cellularassay method for screening ligands against endogenous GPCRs.

Example 4

Dual receptor-specific antagonist screening with the biosensor cellularassays Since the co-stimulation of A431 cells with histamine andepinephrine led to a DMR signal that contains characteristics from boththe histamine- and epinephrine-mediated signaling, the possibility ofdual receptor-specific screening with the biosensor cellular assays wasexamined. This assay proceeds with the initial agonist screening withthe biosensor for one hour, followed by a co-stimulation with histamineand epinephrine. The impacts of compounds in the library on both thehistamine response and the epinephrine were examined based on theircorresponding DMR amplitudes (FIG. 9). For the epinephrine response,there were 77 hits that caused complete inhibition (20±30 pm), 57partial inhibitors (90±40 pm), and 1,146 non-inhibitors (190±60 pm). Forthe histamine response, there were 51 hits that caused completeinhibition (15±50 pm), 79 partial inhibitors (165±100 pm), and 1,160non-inhibitors (370±110 pm).

Correlation analysis between the initial agonist screening and thesubsequent antagonist screening provides further clarification about theaction of positive hits acting on H1R or β2AR. Histamine led to aG_(q)-like DMR signal and also caused the complete desensitization ofcells to histamine but not to epinephrine. All known H1R antagonists inthe library did not lead to any DMR signal. These ligands included(±)-brompheniramine, (±)-chlorpheniramine, (+)-brompheniramine,(+)-chlorpheniramine, clemizole, clemastine, diphenhydramine,fexofenadine, doxylamine, methapyrilene, promethazine, pyrilamine,terfenadine, ketotifen, loratadine, pheniramine, and triprolidine.Except for ketotifen, loratadine and pheniramine that at 1 microM onlycaused partial inhibition of the histamine response, all otherH1R-specific antagonists completely or almost completely attenuated thehistamine response. Conversely, neither H2R- nor H3R-specificantagonists in the library caused any significant inhibition on thehistamine response, suggesting that the histamine response is largelyH1R-specific. Interestingly, the two H2R antagonists famotidine andSKF95282 led to a DMR that was similar to pindolol, and also causedpartial desensitization of cells to epinephrine but not to histamine,suggesting that the two ligands crosstalk to endogenous G_(s)-coupledreceptors in A431. Furthermore, several adrenoceptor uptake/reuptakeinhibitors almost completely inhibited the histamine response, but hadlittle effect on the epinephrine response. They were protriptyline,phenoxybenzamine, amoxapine, maprotiline, desipramine, nortriptyline,amitriptyline, and doxepin.

The adrenergic receptor ligands that led to significant DMR signals alsocaused desensitization of cells responding to epinephrine; and thedesensitization was largely correlated with the amplitude of aligand-induced P-DMR event. However, these ligands greatly differ intheir ability to attenuate the histamine response. Conversely, foradrenergic receptor ligands that did not result in any apparent DMRsignals, only 10 of these antagonists inhibited or partially attenuatedthe epinephrine response. These were alprenolol, betaxolol,S(−)-timolol, (S)-(−)-propafenone, (S)-propranolol, (±)-metoprolol, SR59230A, (±)-propranolol, ICI 118,551, and (±)-Sotalol. The ten betablockers had little impact on the histamine response.

Evidence accumulated in recent years suggest that receptors and theirdownstream signaling pathways do not work in isolation. They areconnected via many fold interactions (cross-talk) and associated insignaling networks. Distinct receptors can cross-talk at multiplelevels. This cross-talk can occur through interaction of intracellularsignal transduction pathways, phosphorylation of receptors andregulatory proteins by kinases, or effects on intracellular calciumrelease. This cross-talk ensures the exchange of information between theindividual signaling pathways and provides the molecular basis for theircooperation. Thus, stimulation of a particular receptor leads toactivation of a signaling pathway that can subsequently interact withthose activated by other receptors. There has been growing recognitionand acceptance that physical interaction between cell surface receptorsmay provide a useful method of accomplishing receptor cross-talkinvestigations. Receptor cross-talk represents a method of fine-tuningthe control of cellular function and is relevant to understandingdisease and response to therapeutic agents that interact withcell-surface receptors. Cross-talk between different G protein-coupledreceptors (GPCRs) is well known and results mostly in synergisticeffects and the amplification of cellular responses. Using the disclosedbiosensor cellular assays, we have found that histamine slightlyattenuated the epinephrine response, whereas epinephrine partiallyattenuated the histamine response, both in a dose-dependent manner. Itis known that the cAMP-PKA pathway plays a central role in theintegration of signaling mediated through distinct classes of GPCRs; andthe ubiquitous second messenger Ca²⁺ can regulate many adenylylcyclases, and such regulation provides an overarching mechanism forintegrating the activities of these two major signaling systems.Furthermore, the co-stimulation DMR signal is largely, but not simply anadditive function of the two individual DMR signals, but also indicatesthat there may be cross-talk between signaling mediated through the tworeceptors. In addition, many adenosine receptor agonists in the libraryalso caused complete attenuation of the epinephrine response. This isconsistent with the well-established heterologous desensitization inwhich distinct G_(s)-coupled receptors can cause cross-desensitizationthrough the cAMP-PKA pathway.

The disclosed biosensor cellular assays can be used for multiplexedscreening in several respects. First, the biosensor cellular assays aremultiplexing in nature for agonist screening. The biosensor is capableof monitoring endogenous receptor activation, leading tohigh-information and physiologically relevant measures of areceptor-ligand pair. These assays do not require prior knowledge ofcell signaling, and are pathway-unbiased. However, the optical responsesrecorded are pathway-sensitive, and do reflect the complexity ofreceptor signaling. A431 cells endogenously express adenosine receptors,and β2AR and histamine receptors, whose ligands are also presented inthe LOPAC library. In this agonist screening, ligands at 1 μM were usedto stimulate the cells. Results showed that ligands that led tosignificant DMR signals were primarily from three families of compounds:ligands for adrenergic receptors; adenosine receptors; and histaminereceptors. Some dopamine receptor ligands that are known to be able toactivate the β2AR also led to an epinephrine-like response, and wereidentified as hits for the β2AR. However, four of the P2Y agonists inthe library did not lead to any significant DMR signals, probably due totheir low potencies (data not shown). Since A431 cells expresses otherreceptors (e.g., protease activated receptors, bradykinin B2 receptor,and epidermal growth factor receptor) whose activation was alsodetectable with the biosensor cellular assays, ligands for thesereceptors if presented in the library would also be identified as hits.

The disclosed biosensor cellular assay methods can also be multiplexingfor antagonist screening. Besides desensitization upon repeatedstimulation with agonists that target the same receptor, in many casescells tend to loss their responsiveness through heterologousdesensitization. Thus, an agonist for another receptor (e.g., adenosinereceptors) could act as an antagonist for the target receptor (e.g.,β2AR). Furthermore, since the biosensor cellular assays measure anintegrated DMR response, an agonist for a receptor (e.g., β2AR) couldpartially attenuate the response of the target receptor which evenmediates signaling through distinct pathway (e.g., a G_(q)-coupled H1R).In addition, pathway modulators can also act as an antagonist for thetarget receptor.

A ligand-induced DMR signal, as measured using the label-free opticalbiosensor, can be an integrated response and consists of contributionsof many cellular events downstream of the receptor activation,particularly those leading to significant mass redistribution within thesensing volume of the biosensor. Contributions from these eventsmediated through a receptor that make the biosensor cellular assays sovaluable, however, also render the optical signal obtained“non-specific” relative to conventional cellular assays. Many ligandsoften exhibit cross-activity to more than one receptor in a cell type orcell system. Furthermore, many GPCR ligands can often induce anoperative bias to activate specific portions of cell signaling through areceptor, and thus exhibit ligand-directed functional selectivity.Therefore, caution should be exercised when analyzing GPCR ligandpharmacology and screening ligands for the target receptors using thebiosensor cellular assays. To achieve target-specific screening, severalapproaches can be applied. For a given cell or cell system, receptorpanning should be performed to determine how many receptors can bedetected using the biosensor cellular assays. Then receptor biology andligand pharmacology should be studied to determine the potential forinterference of a receptor with the target receptor, and tosystematically evaluate the signaling potentials of the target receptor.If one or more endogenous receptors interfere with the target receptor,a cocktail solution containing antagonists that block the activity ofthese receptors can be used to minimize false positives for the targetreceptor. Alternatively, a cell engineering approach can also be used toeither boost the target-specific DMR signal, or to suppress the signalmediated through receptors other than the target. A counter screenbetween a parental cell line and an engineered cell line having or nothaving the target receptor can be performed to establish positive hitsfor the target receptor.

The disclosure has been described with reference to various specificembodiments and techniques. However, it should be understood that manyvariations and modifications are possible while remaining within thespirit and scope of the disclosure.

REFERENCES

-   1. Fang, Y., et. al., J. Biophys. J. 91, 1925-1940(2006).-   2. Fang, Y., et. al., Anal. Chem. 77, 5720-5725(2005).-   3. Fang, Y., et. al., FEBS Lett. 579, 6365-6374(2005).-   4. Fang, Y., et. al., J. Pharmacol. Toxicol. Methods 55,    314-322(2007).-   5. Fang, Y., Assays and Drug development Technologies, 4,    583-595(2006).

1.-13. (canceled)
 14. A label-free screening method comprising:providing an optical biosensor having a live-cell immobilized on asurface of the biosensor, the live-cell having a first target and asecond target; contacting the immobilized cell with a mixture of a firstmarker and a second maker, wherein the first marker activates the firsttarget, and the second marker activates the second target; contactingthe marker mixture-treated immobilized cell with a ligand candidate fora time; and determining the effect of the markers on the ligandcandidate-induced biosensor output.
 15. The method of claim 14 whereinthe first target and the second target are distinguishable from eachother by the biosensor.
 16. A label-free screening method comprising:providing an optical biosensor having a live-cell immobilized on asurface of the biosensor, the live-cell having a first target and asecond target; contacting the immobilized cell with a solutioncontaining at least one blocker; contacting the immobilized cell with aligand candidate; contacting the ligand candidate-treated cell with amixture containing, a first and a second marker, wherein the firstmarker activates the first target, and the second marker activates thesecond target; and determining the effect of the ligand candidate on themarker mixture-induced biosensor output.
 17. The method of claim 16wherein the blocker comprises at least one of an antagonist, aninhibitor, an interference RNA, an anti-sense nucleic acid, or aninhibitory antibody for a cellular protein that is not themarker-intended target, but whose activation interferes with thebiosensor response of the targets.
 18. The method of claim 16 whereinthe blocker is added before the ligand candidate.
 19. The method ofclaim 16 wherein the blocker is added together with the ligandcandidate.
 20. A label-free screening method comprising: providing anoptical biosensor having a mixed population of cells containing twotypes of cells, a first type of cells expressing a first target and asecond type of cells expressing a second target, wherein the two typesof cells are co-immobilized on a surface of the biosensor; contactingthe immobilized cells with a mixture containing two markers, wherein thefirst marker activates the first target in the first type of cells, andthe second marker activates the second target in the second type ofcells; contacting the mixture-contacted cells with a ligand candidate;and determining the effect of the markers on the ligandcandidate-induced biosensor output.
 21. The method of claim 20 whereinthe two types of cells comprise: a parental cell and an engineered cellexpressing a target; two engineered cells each expressing a target; ortwo native cells.
 22. A label-free screening method comprising:providing an optical biosensor having a mixed population of cellscontaining a first type of cells expressing a first target and a secondtype of cells expressing a second target, both cell types areco-immobilized on a surface of the biosensor; contacting the immobilizedcells with a ligand candidate; contacting the ligand candidate-treatedcells with a mixture containing two markers, wherein the first markeractivates the first target in the first type of cells, and the secondmarker activates the second target in the second type of cells; anddetermining the effect of the ligand candidate on the markermixture-induced biosensor output.